The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and be able to be easily automated. Sensitivity, i.e. detection limits, remains a significant obstacle in nucleic acid detection systems, in particular where only small-volume and/or low concentration samples are available. A variety of techniques have been developed to address this issue including analyte amplification. Analyte amplification involves the amplification (i.e. replication) of the analyte sequence being detected in a sample, resulting in a significant increase in the number of analyte molecules. The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies, which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis.
In their standard format, amplification technologies for nucleic acid analysis have a number of limitations to their commercial utilization including the cost of the reagents and the ability to automate the process.
In microarray technology, varied and important information is generated and many potential applications are provided. The use of these microarrays in research, diagnostic and related applications has grown considerably and is expected to continue to do so. A variety of different array technologies have been developed in order to meet the growing need of the biotechnology industry, as evidenced by the extensive number of patents and other literature published.
In view of the micro formats of the samples that are used for microarray analysis, amplification of the analyte molecules is certainly desired. The feasibility of miniaturized amplification reactions was demonstrated in US 2002/0037510 A1 wherein amplification reactions performed within micro spots arrayed on a glass slide are disclosed.
However, as well appreciated in the art, there is a continuous and expanding need for improved microarray analysis methods securing rapid, highly specific methods of identifying and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures.
It is an object of the present invention to provide improved detection limits and time management practices in automated microarray analysis systems. It is an object of the present invention to provide high-throughput microarray analysis methods with integrated amplification-hybridization-detection of sample analytes. As such, in addition to large scale miniaturized sensitive analysis practices, the present invention provides a parallel amplification/identification of analyte molecules in a sample, thereby further integrating an efficient direct detection of the identified amplified sample analytes. Any time-consuming step of e.g. a pre-analysis amplification step and/or washing off excess label for detection is omitted.